It is recently discovered that a significant portion of the modified cytosines in mammalian genomes is 5-hydroxymethylcytosine (5-hmC), an oxidation product of 5-methylcytosine (5-mC). Current methods including bisulfite conversion cannot distinguish or determine its genomic locations. The proposed research in this grant application is based on a novel family of modification-dependent restriction endonucleases (REs), represented by PvuRts1I. Unlike other existing REs, these enzymes recognize 5-hmC in DNA and cleave at fixed distances away from their recognition sites. The glucosylation status of the 5-hmC can have significant effects on the cleavage efficiency. Using ultra high throughput sequencing platforms and genomic DNA digested with PvuRts1I family enzymes, one should be able to identify and map 5-hmC reliably. Therefore, application of these PvuRts1I family enzymes can provide a foundation for the next generation of methods for analyzing epigenetic modification. In Phase I research, we plan to purify the recombinant enzymes and characterize their biochemical properties in detail in vitro. In Phase II research, we plan to determine the molecular structure of at least one of these enzymes both in its apo-form (without DNA) and as an enzyme complex with a 5-hmC DNA substrate. We will establish methodologies whereby these enzymes can be used to decode the DNA hydroxymethylation patterns in human, mouse, and several other model organisms. We will also examine the dynamics of DNA hydroxymethylation during mouse embryonic stem cell differentiation and at various developmental stages. Another goal of the Phase II research will be to isolate mutants that will contain improved properties. This work will be based on the molecular structures and our previously established enzyme engineering protocols. Furthermore, emphasis will be given to isolating mutants that have no enzymatic activity, yet have high binding affinity for the 5-hmC to be used as an affinity reagent and for in vivo labeling of the 5-hmC in mammalian nuclei. We believe our proposed research is innovative and timely, and will help to decode the next layer of epigenetic information in the mammalian genome. A full understanding of these enzymes and their novel applications in decoding epigenetic information will allow us to develop new products and kits that should have a major impact for the broader biomedical community interested in studying epigenetic modifications.